The long-term goal of this project is to advance our knowledge of the human beta cell by providing a detailed understanding of the 3D biology of its normal environment - the human pancreatic islet - through comparative high-resolution three-dimensional (3D) structure studies of islets of Langerhans isolated from mouse and human pancreas. Using technology developed for the improved ultrastructural preservation and imaging of immortalized (rodent) beta cell lines and isolated rodent islets as its foundation, this application seeks funds for additional personnel to assist with the substantial task of optimizing these methods further for successful application to human tissue. Specifically, this technology combines methods for (1) cooling intact islets to -196 degrees C within approximately 10 milliseconds by high-pressure freezing to "capture" islet/beta cell physiology in a vitrified, close-to-native state, (2) ultralow temperature tissue processing/fixation (freeze-substitution), and (3) high resolution (<=5nm) electron microscope (EM) tomography. This powerful combination of state-of-the-art structural methods has already provided important new insights into the mechanisms and machinery involved in the synthesis, processing and secretion of insulin in glucose-stimulated beta cells in rodent cell culture models and tissue. If funded, this application will allow us to address a number of significant technical challenges associated with the use of these methods with human tissue. Moreover, this work will pave the way for more detailed scientific investigations aimed at determining how the 3D biology of the human islet/beta cell relates to the development, physiology and dysfunction of the human endocrine pancreas in the near future.